Recent work has centered on the study of the mechanism(s) by which cholera toxin secreted from the cytosol of Vibrio cholerae, assembled into holotoxin, and released from the bacterial cell. We have found that a 45,000 molecular weight polypeptide is synthesized in vitro in Escherichia coli extracts programmed with free polysomes extracted from Vibrio cholerae 569B. A similar size polypeptide has been shown to accumulate in the outer membrane of V. cholerae grown in the presence of subinhibitory concentrations of phenethyl alcohol, as well as in the membrane fraction of one class of hypotoxinogenic mutant of V. cholerae 569B. We are currently testing the hypothesis that cholera toxin is synthesized on free polysomes and is secreted to the outer membrane, perhaps through Bayer's junctions, where it is processed into subunits, assembled into holotoxin, and then released from the bacterial cell.